Our long-range goal is to generate a mechanistic picture of clathrin-based membrane vesicular traffic - a """"""""molecular movie"""""""". To produce this movie, we will integrate results from studies in vitro of protein-protein recognition and assembly, studies using x-ray crystallography and electron microscopy of components of the endocytic molecular machinery, and studies in living cells of temporal sequences of protein associations and translocations. We have dedicated much of the grant period just ending to analyzing clathrin structure and assembly, to studying cargo recognition and recruitment, and to developing light-microscopic techniques that will allow us to study the clathrin pathway in living cells. During the coming grant period, we will extend the scope of our inquiry to other steps in the formation and targeting of clathrin-coated vesicles and to connect molecular structures and intracellular dynamics. We will link this description of coated-vesicle dynamics to molecular structural studies of uncoating (Aim 1), of adaptor function (Aim 2), and to cell-biological studies linking endocytosis and the actin cytoskeleton (Aim 3).
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